ABSTRACT: Cell viability requires maintenance of genome integrity, which is continuously threatened by intracellular and external sources of DNA damage. One prevalent source of DNA damage is UV light, which generates cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone (6-4 PP) adducts. The nucleotide excision repair (NER) pathway is mainly responsible for the removal and repair of these lesions. Two different branches of the NER pathway contribute to repair: transcription coupled repair (TCR) recognizes CPDs at transcriptionally active loci, while global genomic repair (GGR) marks lesions in other parts of the genome. Trans-lesion synthesis (TLS) operates in parallel to bypass any unrepaired lesions in S phase. Mutations in many of these components cause photosensitivity diseases, notably Xeroderma Pigmentosa and Cockayne Syndrome. Recently, we have identified methylation of RNA at the 6 position of adenosines (6mA) as an exciting new contributor to DNA repair. 6mA RNA is rapidly and transiently induced at damage sites following UV exposure, and we have identified the RNA methyltransferase METTL3 as being responsible for this modification. Our preliminary results suggest that METTL3 may participate in the TCR branch of the NER pathway, as repair of transcribed regions is specifically compromised in METTL3 knock-out (KO) cells. Accordingly, loss of 6mA RNA by METTL3 KO decreased cell survival after UV exposure, which was rescued by wild-type, but not catalytically-inactive, METTL3. The goals of this work are to determine the role and mechanism of action of 6mA in DNA repair. We will determine whether 6mA RNA functions to repair transcribed and/ or non-transcribed regions genome-wide, and identify in which pathways/ steps of NER it participates. We will also explore its role in TLS and repair of other types of DNA damage to determine how broadly 6mA RNA operates in the DNA damage response (DDR). Secondly, we will investigate the hypotheses that 6mA marks transcripts arising from damaged DNA templates for degradation, and/ or that 6mA influences alternative splicing or translation of modified transcripts, particularly of those encoding proteins important for DNA repair or cell survival. Finally, we will identify and characterize reader proteins that recognize 6mA RNA, in order to investigate the hypothesis that these readers play a mechanistic role in linking 6mA RNA to the DDR. Our discovery of an RNA modification mediating DNA repair has uncovered a new and exciting facet to the DDR and to our knowledge of how cells maintain genome integrity. The research proposed in this application will provide important new insight into how 6mA RNA regulates the DDR, and our findings will be important for understanding and treating photosensitivity diseases.